Errors in chromosome segregation cause birth defects and genetic instability in tumor cells. The spindle checkpoint is a control circuit that reduces these errors by keeping cells from starting chromosome segregation until all their chromosomes have been properly aligned on the mitotic spindle. This application proposes genetic and cell biological studies of the spindle checkpoint in budding yeast to ask how cells minimize errors in chromosome segregation. The experiments will combine genetic manipulations that alter the number, structure, and environment of the centromeres with sophisticated microscopy that monitors the behavior of chromosomes in living cells. Experiments are proposed to: 1) Use natural and synthetic kinetochores to dissect the interaction of chromosomes and microtubules that create the mitotic spindle. Controlling the number and nature of functional kinetochores will reveal what determines the number of microtubules in the spindle, the length of the spindle in metaphase, the rate at which kinetochores attach to and detach from spindle microtubules, and the activity of the spindle checkpoint. 2) Create, characterize, and exploit synthetic activation of the spindle checkpoint. High local concentrations of checkpoint proteins activate the checkpoint. These reactions will be investigated and used to create the minimal reactions needed to activate the checkpoint in vivo and in vitro. 3) Test a model for checkpoint activation by asking if the distance between sister kinetochores regulates the local concentration of Ipl1 (an Aurora B homolog) at the kinetochore, thus monitoring tension at the kinetochore. This idea will be tested by independently regulating the stretch between sister kinetochores and the concentration of Ipl1 at the kinetochore. 4) Develop a yeast model for the generation of genetic instability during cancer progression. Genetic instability is universal in human cancer. Mutations in many genes, including spindle checkpoint proteins, can cause genetic instability, but the cause of instability in most patients is obscure. Yeast strains will be used to select for the inactivation of multiple growth suppressor genes, mimicking the inactivation of multiple tumor suppressor genes in cancer. These strains will reveal how genome architecture influences how fast genetic instability spreads through a population and identify the mutations that give rise to instability.